CN102268390A - Bacillus amyloliquefaciens for generating fungi apoptosis lipopeptide, and preparation method and application thereof - Google Patents
Bacillus amyloliquefaciens for generating fungi apoptosis lipopeptide, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses Bacillus amyloliquefaciens for generating fungi apoptosis lipopeptide, and a preparation method and application thereof. The preparation method comprises the following steps of: A, screening and separating Bacillus amyloliquefaciens, namely acquiring rice in a tillering stage, washing the root of the rice, soaking in ethanol for disinfection, dicing the root of the rice, and culturing on a culture medium to obtain a mono-colony; B, detecting antibacterial activity of bacteria, namely culturing the separated bacteria by using a PD liquid, and detecting the antibacterial activity; C, classifying and identifying the bacteria, namely performing morphology identification on the detected bacteria with obvious antifungal activity; and D, culturing and storing the bacteria, namely culturing the Bacillus amyloliquefaciens on a PD culture medium, and storing the strains at low temperature. The Bacillus amyloliquefaciens is applied to preparing medicines for treating or preventing oil camelliae anthracnose; the method is practicable, and easy and convenient to implement; the lipopeptide has wide antibacterial spectrum, high antibacterial activity, high stability and stable control effect; and an antibacterial substance generated by the Bacillus amyloliquefaciens, namely the lipopeptide has stable activity, high temperature resistance and proteolysis resistance, and is a high-efficiency and stable antibacterial substance.
Description
Technical field
The invention belongs to technical field of agricultural microbiology, be specifically related to a kind of bacillus amyloliquefaciens of fungus apoptosis lipopeptide, the preparation method who also relates to a kind of bacillus amyloliquefaciens of fungus apoptosis lipopeptide simultaneously also relates to a kind of purposes of bacillus amyloliquefaciens of fungus apoptosis lipopeptide.
Background technology
A lot of bacilluss can suppress fungi growth by producing lipopeptid.Why lipopeptid can suppress fungi growth, is because its amphipathic structures shape, and lipopeptid interacts with cytolemma and can cause perforate membrane to cause protoplasma to reveal, and makes mycelium rupture; Lipopeptid can also pass through dissolving pathogenic bacteria archespore wall or cytolemma, thereby causes cell wall perforation, deformity etc. to suppress spore germinations, the document that sees reference (Qi Gaofu; Zhao Xiuyun; Zhu Fayin; Wen Kai; Zhang Shichao; Explain sub-ox.The bacillus amyloliquefaciens of short fungus apoptosis lipopeptide and lipopeptid and the application that is prepared by this bacterium are produced in one strain.The patent No.: CN200810197403.4).
Applicant unit one belongs to is separated to a strain bacillus amyloliquefaciens CH1 from rice root, can produce lipopeptid class surfactivity element by fermentation, this lipopeptid has significant antagonistic effect to various plants pathogenic fungies such as Rhizoctonia solani Kuhn, southern corn leaf blight, cotton-wilt fusarium, wheat scab, cotton anthracnose bacterium, dothiorella gregaria bacterium, botrytis cinereas.Further discover, this lipopeptid not only comprises the restraining effect principle of fungi necroses hypha,hyphae, and be included in concentration and induce fungi that apoptosis takes place when low, document (Qi sees reference, G.F., Zhu, F.Y., Du, P, et al.Lipopeptide induces apoptosis in fungal cells by a mitochondria-dependent pathway.020Peptides, 2010,31:1978-1986 and Lin Fucheng, Li Debao, subtilis (Bacillus subtilis) S9 is to the bacteriolysis of plant pathogenic fungi, Plant Pathology, 2003,33:174-177).
On slide glass, inoculate the experiment that stands facing each other of CH1 and Rhizoctonia solani Kuhn respectively, found that tangible phenomena of apoptosis has appearred in the Rhizoctonia solani Kuhn that is suppressed on slide glass, but do not detect corresponding necrocytosis feature, the antagonistic bacterium of supposition bacillus class under physical environment, as Bacillus subtillis, bacillus licheniformis, bacillus amyloliquefaciens etc., the lipopeptid concentration deficiency that produces is so that the pathogenic fungi necrosis normally suppresses the growth of pathogenic fungi by the form of inducing fungal cell's apoptosis.
Current, the use of chemical pesticide has caused serious ecological damage and environmental pollution in the whole world, and using the biological pesticide of high-efficiency low-toxicity is the inexorable trend of agricultural development.Fermentation optimization to CH1 in this experiment at first adopts the Plackett-Burman design to filter out the factor that lipopeptid output is had remarkably influenced, has obtained the optimum combination of each component of substratum then with the design of field mouth.The fermented liquid aftertreatment adopts the spraying drying mode to carry out, and in the spraying drying, high pressure draft makes fermented liquid form ultra-fine drop, makes the instantaneous drying of fermented liquid, has reduced the injury of high temperature to activeconstituents in the fermented liquid to greatest extent, has kept bacteriostatic activity.
This research also detects by nursery, the field experiment drug effect to the CH1 powder, and compares with the common chemical sterilant, for the research and the use of lipopeptid class biological pesticide provides reference.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, the objective of the invention is to be to provide a kind of bacillus amyloliquefaciens of fungus apoptosis lipopeptide, this bacterium can produce a kind of lipopeptid of short fungi apoptosis, can be used as the effective constituent of anti-phytopathogen.This bacterium is to Rhizoctonia solani, Helminthosporium maydis, Fusarium oxysporium, Botrytis cinereapers, Gibberella zeae, Dothiorella gregaria, plant pathogenic fungis such as Colletotrichum gossypii all have tangible antibacterial effect.It is remarkable that fermented liquid suppresses effect to oil tea anthrax bacteria (Colletotrichum gloeosporioides).
Another object of the present invention is the preparation method who has been to provide a kind of bacillus amyloliquefaciens of fungus apoptosis lipopeptide, the post-treating method of fermention medium, fermentation condition and the fermented liquid of this bacterium is groped and optimized, fermented liquid is carried out low pressure concentrate and the spraying drying aftertreatment, the powder drug effect rate of retaining that obtains after the spraying drying is 86.2%.This pulvis is easy to use, but standing storage in room temperature, efficacy stability is converted water dilution back spray application, can be used for fungal diseases such as field large area control oil tea anthrax.
One purpose in addition of the present invention is the application of bacillus amyloliquefaciens in preparation treatment or prevention oil tea anthrax medicine that has been to provide a kind of fungus apoptosis lipopeptide.The nursery experiment showed, that spray-dried powders can be good at preventing and treating oil tea anthrax.Be used for the experiment of nursery, field behind 1000 times of the Dilution for powder, control oil tea anthrax bacteria, the preventive effect of bacillus amyloliquefaciens (B.amyloliquefaciens) CH1 pulvis is higher than the common chemical agricultural chemicals, and preventive effect is up to 74.4%; It only is 48.7% that 70% thiophanate methyl wettable powder, preventive effect reach 59.0%, 50% carbendazol wettable powder.Show that this bacterium can effectively prevent and treat the oil tea anthrax bacteria, and compare that environmentally safe can not damage people and animals with chemical pesticide.This bacterium can be used for preventing and treating oil tea anthrax, helps producing high-quality nuisance-free plant oil.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
A kind of preparation method of bacillus amyloliquefaciens of fungus apoptosis lipopeptide the steps include:
1. the screening and separating of bacillus amyloliquefaciens (Bacillus amyloliquefaciens): from rice terrace gather tillering phase spend 11 paddy rice, the rice root outside surface is rinsed well with tap water, again with aseptic water washing 2-4 time, then with 70% (v/v) alcohol immersion sterilization 28-32s, behind aseptic water washing 2-4 time, rice root is cut into the fragment of about 1mm size, place the PDA substratum (to contain the 200g potato and cook liquid, 20g glucose, 20g agar, adding distil water is to 1L) go up in 28 ℃ of cultivation 46-50h, obtain single bacterium colony.
2. the bacteriostatic activity of bacterial detection: the bacterium that separation is obtained (contains the 200g potato and cooks liquid with the PD liquid nutrient medium, 20g glucose, adding distil water is to 1L) in 28 ℃, shaking culture, fermented supernatant fluid with 0.22 μ m membrane filtration after, mix the Rhizoctonia solani Kuhn of falling the plating (Hua Zhong Agriculture University's plant protection is a plant pathology teaching and research room) by volume with the PDA substratum, document (cloud monthly interest sees reference, champion Xu. Cantharidin is to the inhibiting research of phytopathogen. Hubei University's journal: natural science edition, 2003,25 (4): 342-345), detect anti-microbial activity.
3. the classification of bacterium is identified: the bacterium that detects remarkable anti-mycotic activity is carried out 16SrRNA and identification of morphology.Extract the genomic dna of bacterium with ordinary method, and the document that sees reference (work such as Sa nurse Brooker, Jin Dongyan etc. translate. molecular cloning experiment guide second edition. and Beijing: Science Press, 1999), carry out PCR with the universal primer of 16SrRNA.Amplified production carries out being defined as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) through the Blast comparative analysis behind the sequencing in NCBI.
4. microbial culture and store method: the condition of cultivating bacillus amyloliquefaciens is PD substratum or LB substratum, 28 ℃ of-37 ℃ of cultivations.The bacterial strain store method: get cultured Bacillus.amyloliquefaciens CH1 bacterium liquid add sterile glycerol to final concentration 25% in-80 ℃ of preservations.
The applicant carries out fermentation optimization by the strain bacillus amyloliquefaciens (deposit number is CCTCC NO:M208127) to Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province and fermentation is amplified, and fermented liquid carried out low pressure concentrates and the spraying drying aftertreatment, find to have kept most of bacteriostatic activity after the spraying drying, the nursery experiment showed, that spray-dried powders can be good at preventing and treating oil tea anthrax.The bacillus amyloliquefaciens bacterial strain of the short fungus apoptosis lipopeptide of the isolating product of one strain, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CH1, be deposited in Chinese typical culture collection center (CCTCC), deposit number is CCTCC:M208127, depositary institution address: China. Wuhan. Wuhan University, preservation date: on September 5th, 2008.
The mycology feature of bacillus amyloliquefaciens CH1 (Bacillus.amyloliquefaciens CH1):
Bacillus amyloliquefaciens CH1 Gram-positive, thalline is shaft-like, and thalline size 1-1.4 μ m * 2.3-3.5 μ m (contains the 200g potato and cooks liquid at PDA, 20g glucose, 20g agar, adding distil water is to 1L) can rapid diffusion on the flat board, do not form specific colonial morphology, the edge is irregular, be canescence, thickness has pod membrane, and cultivating the 48h rear surface has little gauffer.On the LB flat board, form the bacterium colony of rice white, colony diameter 3-5mm, surface drying has gauffer, and the centre subsides, and the edge is wavy.37 ℃ of growths are obviously faster than 28 ℃.Brood cell's near-end is given birth to, flagellum Zhousheng.The various plants pathogenic bacteria there is remarkable bacteriostatic activity, bacteriostasis rate when detecting 72h, bacteriostasis rate to rape ash arrhizus bacteria (Botryotini fuckeliana) is 96.9%, (Colletotrichum gloeosporioides) is 77.0% to the pepper anthracnose bacterium, (Cytospora chrysosperma) is 57.0% to willow bark rot bacterium, (Thielaviopsis paradoxa) is 65.1% to sugarcane pineapple germ, (Dothiorella gregaria) is 62.0% to the ring rot of apple bacterium, (Fusarium graminearum) is 65.8% to fusarium graminearum, (Sclerotinia sclerotiorum) is 85.3% to the rape sclerotinite, (Bipolaris maydis) is 93.4% to southern corn leaf blight, and (Colletotrichum gloeosporioides Penz.) is 100% to the oil tea anthrax bacteria.The CH1 nutrient solution of PDB and dregs of beans culture medium culturing all has fungistatic effect preferably to phytopathogen, but 2% dregs of beans substratum (20g bean cake powder, distilled water is settled to 1L) fungistatic effect of tunning obviously is better than PDB substratum (potato 200g, sucrose 20g, distilled water is settled to 1L) tunning.
Bacillus amyloliquefaciens CH1 can produce a kind of ring-type lipopeptid of Surfactin class, called after WH1fungin, lipopeptid can obviously suppress fungal cell's glucan synthase activity, after WH1fungin handles, some cell of Rhizoctonia solani Kuhn and candiyeast can be dyeed by AZO-blue, shows phenomenons such as membrane perforation, tenuigenin seepage.The lipopeptid sequence is as follows:
The application of a kind of bacillus amyloliquefaciens of fungus apoptosis lipopeptide in preparation treatment or prevention oil tea anthrax medicine, its applying step is:
1.CH1 fermentation optimization: with the bacteriostasis rate to oil tea anthrax is respective amount, design the factor that in the medium component that obtains being screened respective amount is had remarkably influenced by Plackett-Burman, by experiment of single factor the factor that obtains is optimized respectively then, obtains the optimum level combination of each factor of substratum at last by the design of field mouth.
2. fermentation is amplified and the fermented liquid aftertreatment: completed successfully 250mL and shaken the amplification of bottle to the 500L jar.To fermented liquid concentrate and spraying drying after the powder drug effect rate of retaining that obtains be 86.2%.
3.CH1 the preparation of spray-dried powders: the amplification of fermenting process is carried out in 3L, 20L, 500L fermentor tank successively, the initial revolution 200rpm that stirs, 30 ℃ of culture temperature, after fermentation is finished, with the concentrating under reduced pressure jar through 65 ℃, 0.09MPa fermented liquid is concentrated, carry out spray drying treatment with spray-drying tower then, obtain spray-dried powders.
4.CH1 the resistance to oil tea anthrax: the applicant finds that by dull and stereotyped inhibition test the CH1 fermented liquid can make oil tea anthrax mycelia deformity, thereby suppresses the growth of oil tea anthrax.Excised leaf control experiment shows that the fermented liquid that is diluted to 50 times of original volumes is to the anthrax preventive effect on the oil tea blade that exsomatizes still highly significant.
5. compared CH1 pulvis and two kinds of chemical bactericides commonly used preventive effect by nursery, field experiment: 1000 times of bacillus amyloliquefaciens CH1 wettable powder dilute with waters to oil tea anthrax, in the processing of spraying of oil tea their early stage, carry out dispenser once every 10d later on, continuous use 5 times.Carry out the investigation first time behind medication 15d, 10d carries out the investigation second time after the last medication.Investigation sickness rate and disease index.
Result of implementation:
Lipopeptid in the CH1 fermented liquid can make oil tea anthrax mycelia deformity, thereby suppresses the growth of oil tea anthrax, sprays the fermented liquid of dilution on excised leaf, can effectively suppress spreading of oil tea anthrax bacteria spot equally, and the protection oil tea is not by infection process.
The result of fermentation optimization: Plackett-Burman result's demonstration, in P＜0.05 scope, bean cake powder, NH
4NO
3, Na
2HPO
4With temperature be the principal element that influences lipopeptid output because temperature is negatively influencing to lipopeptid output, so leavening temperature is chosen 30 ℃, other 3 substratum factors obtain optimal concentration by experiment of single factor and are respectively Na
2HPO
44g/L, NH
4NO
34g/L, bean cake powder 12g/L; Determine that final substratum is combined as bean cake powder 12g/L, NH in conjunction with factors such as field mouth design result and production costs at last
4NO
32g/L, Na
2HPO
42g/L, starch 15g/L.
Fermentation is amplified and the aftertreatment result: in the fermentation amplification process, bacteriostatic activity reduces by 30%.Spray-dired powder finished product output is 6.5g/L, and dull and stereotyped inhibition experiment shows that the pulvis drug effect is 86.2% with the spraying drying primary fermentation liquid phase ratio rate of retaining, and powder normal temperature is preserved active no any reduction after a year.Spraying drying gained powder solubility is good, and stable in properties, but prolonged preservation have kept the benefit born of the same parents of sprouting simultaneously, can be in the dispenser process directly tieback strengthen drug effect to the oil tea blade and in the soil.
The nursery experimental result: investigation result shows for the first time, the preventive effect of bacillus amyloliquefaciens (B.amyloliquefaciens) CH1 pulvis is best, up to 74.4%, next is 70% thiophanate methyl wettable powder, preventive effect reaches 59.0%, 50% carbendazol wettable powder effect is the poorest, and preventive effect only is 48.7%; Investigation result shows for the second time, and CH1 pulvis preventive effect is still leading, is 53.5%, secondly is 70% thiophanate methyl, and preventive effect is that 44.2%, 50% carbendazol wettable powder effect is the poorest, and preventive effect only is 39.5%.
The present invention compared with prior art has the following advantages and effect:
Oil tea anthrax is the main disease on the oil tea, is in a bad way over the years, and the morbidity scope is wide, has significantly reduced oil tea output, has endangered tea grower's interests.For the research of oil tea anthrax Prevention Technique, still mainly be to adopt agricultural and chemical agent control at present, also be in the starting stage for oil tea anthrax biological control technical study.
In the production, the main chemical pesticide (m-tetrachlorophthalodinitrile, Bordeaux mixture, thiophanate methyl, Tuzet, tricyclazole etc.) that uses prevents spreading of oil tea anthrax, yet chemical prevention causes the resistance of germ easily, also strengthened simultaneously pollution, be unfavorable for producing high-quality nuisance-free plant oil oil tea fruit and environment.The life-time service sterilant has caused a series of serious consequences, as contaminate environment, harm humans health, the destruction eubiosis and the increase of pathogenic agent resistance etc.The drug residue problem that chemical pesticide brings is brought up to a new height to the people's diet health problem, so biological green agricultural chemicals nuisanceless, environmental protection more and more is subjected to people's attention.Constantly seek low toxicity efficiently biological pesticide be the extremely urgent task of agriculture researcher.The main component of biogenic pesticide is microorganism itself and its meta-bolites, and these effective constituents generally all have the advantage of degrading easily at occurring in nature, can not damage people and animals.
Bacillus is to use one of the most successful microbial bactericide and sterilant at present, and the antibacterial substance kind that bacillus produces is many, and antimicrobial spectrum is wide, is the main force of antagonism pathogenetic bacteria and pathogenic fungi always.The antimycotic material that bacillus produces is mainly secondary metabolite lipopeptid and gene coded protein.Lipopeptid is developed widely with its has a broad antifungal spectrum, anti-microbial effect advantage strong, good stability.Lipopeptide compound is a type biological surfactant that is produced by the bacillus metabolism.Its structure generally be by 1 beta-hydroxy fatty acid and 7-10 amino acid with the ring that the amido linkage form is connected, have characteristic antimycotic, antitumor, antiviral, mycoplasma, have that the potential plant fungal disease resistance is worth and medical use value.Why lipopeptid can suppress fungi growth, is because its amphipathic structure causes protoplasma to reveal with the cytolemma interaction, makes mycelium rupture; Or the antimicrobial substance that produces is by dissolving pathogenic bacteria archespore wall or cytolemma, thereby causes phenomenons such as cell wall perforation, deformity to suppress spore germinations.
Be separated to a kind of bacillus amyloliquefaciens CH1 from rice root, can suppress fungi growth by producing the surfactant-based WH1 fungin of lipopeptid class.Why WH1 fungin can suppress fungi efficiently, be because it can be in different concentration, bring into play anti-microbial effect by different mechanism: when high density, the inducing cell perforate membrane, cause tenuigenin to be revealed, cause the fungal cell to break and death, and when lower concentration can procedural apoptosis take place by inducing the fungal cell, make fungi nuclear fragmentation occur, the ROS accumulation, phosphatidylserine turns up, typical apoptosis feature such as caspase enzymic activity rising, and this mode may be produce lipopeptid class bacillus and suppress the main mode of fungi at occurring in nature, and document sees reference.Bacillus amyloliquefaciens CH1 is the bacillus of report energy high-efficiency prevention and control oil tea anthrax, and its prevention effect is stable, is better than common chemical sterilant (methyl holder, derosal).
And bacillus amyloliquefaciens CH1 has good prevention effect to oil tea anthrax, the potential biological pesticide that is developed to control oil tea anthrax, and fungistatic effect and the fermentation manufacturing technique of CH1 carried out further exploration, by substratum and Optimizing Conditions of Fermentation, improve the output of antibacterial lipopeptid, and tunning is further concentrated and spray drying treatment, but with as the convenient transportation prolonged preservation, specially at the wettable powder of oil tea anthrax.Spray-dried powders bacteria containing amount height reaches 0.5 hundred million brood cell/grams, but unsuitable environmental conditions such as brood cell's withstand high temperatures, drying, radiation, the antimicrobial substance-lipopeptid of its generation is activity stabilized, and high temperature resistance, proteolysis resistant are a kind of antimicrobial substances of efficient stable.The trend of developing green emerging biological pesticide has been complied with in this research on the one hand, also will inject new strength for the biological pesticide exploitation of China on the other hand.
Description of drawings
Fig. 1 is the restraining effect synoptic diagram of a kind of bacillus amyloliquefaciens to oil tea anthrax.
Figure 1A: on the PDA flat board, the grow state of 24h of oil tea anthrax spore;
Figure 1B, C, D: on the PDA flat board that contains 1/50,1/20,1/10 fermented liquid, the grow state of 24h of oil tea anthrax spore.In substratum, add the growth that the CH1 fermented liquid can suppress oil tea anthrax bacteria silk significantly.
Fig. 2 is the teratogenesis of a kind of lipopeptid of bacillus amyloliquefaciens generation to oil tea anthrax mycelia.
Fig. 2 A: the oil tea anthrax mycelia that contains growth 24h on the PDA flat board of 1/10 fermented liquid;
Fig. 2 B: the oil tea anthrax mycelia of growth 24h on the PDA flat board.Lipopeptid can make oil tea anthrax mycelia expand deformity as we can see from the figure, thereby suppresses its growth.
Fig. 3 is a kind of bacillus amyloliquefaciens CH1 prevention effect synoptic diagram to oil tea anthrax on the oil tea excised leaf.
Fig. 3 A inserts oil tea anthrax for after spraying the CH1 fermented liquid that dilutes 50 times, cultivates the 5d situation; Fig. 3 B inserts oil tea anthrax for after spraying distilled water, cultivates the situation of 5d.Can see that CH1 can suppress the diffusion of oil tea anthrax on blade more significantly.
Embodiment
Embodiment 1:
A kind of preparation method of bacillus amyloliquefaciens of fungus apoptosis lipopeptide the steps include:
1, the screening and separating of bacillus amyloliquefaciens (Bacillus amyloliquefaciens):
From Chinese Wuhan City, Hubei Province Hua Zhong Agriculture University rice terrace get tillering phase spend 11 paddy rice to count strain, clean the back and separate root.After the rice root outside surface rinsed well with tap water, again with aseptic water washing three times, then with 70% (v/v) alcohol immersion sterilization 30s, behind aseptic water washing 3 times, with sterile scissors rice root is cut into the fragment of about 1mm size, places the PDA substratum (to contain the 200g potato and cook liquid, 20g glucose then, 20g agar, adding distil water is to 1L) go up in 28 ℃ and cultivate 48h.The bacterium that separation is obtained (contains the 200g potato and cooks liquid with the PD liquid nutrient medium, 20g glucose, adding distil water is to 1L) in 28 ℃, shaking culture 48h in the 180rpm shaking table, centrifugal, collect supernatant liquor and with behind the 0.22 μ m membrane filtration, be respectively 10% by volume with the PDA substratum, 5%, 2.5% mixes the Rhizoctonia solani Kuhn of falling the plating (Hua Zhong Agriculture University's plant protection is a plant pathology teaching and research room), the document that sees reference (cloud monthly interest, champion Xu. Cantharidin is to the inhibiting research of phytopathogen. Hubei University's journal: natural science edition, 2003,25 (4): 342-345), detect anti-microbial activity.
2, the classification of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is identified:
The bacterium that detects remarkable anti-mycotic activity is carried out 16SrRNA and identification of morphology.The bacterium that is separated to PD liquid nutrient medium incubated overnight, extract the genomic dna of bacterium, the document that sees reference (Qi, G.F. with ordinary method, Zhu, F.Y., Du, P., et al.Lipopeptide induces apoptosis in fungal cells by a mitochondria-dependent pathway.Peptides, 2010,31:1978-1986), to measure the universal primer (primerF:AGAGTTTGATCCTGGCTCAG of 16SrRNA; PrimerR:AAGGAGGTGATCCAGCCGCA, giving birth to worker's biotechnology company limited by Shanghai synthesizes) (the PCR reaction system is: distilled water 18.3 μ l to carry out PCR, 10 * PCR damping fluid, 2.5 μ l, general positive anti-primer, each 1 μ l of dNTP, Taq enzyme 0.2 μ l (the PCR reaction reagent is east, Guangdong and contains the bio tech ltd product).Response procedures: 94 ℃ of 4min, 94 ℃ of 45s of 30 round-robin, 52 ℃ of 45s, 72 ℃ of 90s, 72 ℃ of 10min again), 1% agarose electrophoresis detects the dna fragmentation that the size that successfully increases is approximately 1.5kb, gives birth to worker's biotechnology company limited by Shanghai and carries out being defined as bacillus amyloliquefaciens (Bacillus.amyloliquefaciens) through the Blast comparative analysis behind the sequencing in NCBI.
The condition of cultivating bacillus amyloliquefaciens is PD substratum or LB substratum, 28 ℃ of-37 ℃ of cultivations.The bacterial strain store method: get cultured Bacillus.amyloliquefaciens CH1 bacterium liquid add sterile glycerol to final concentration 25% in-80 ℃ of preservations.
3, the fermentation condition optimization of bacillus amyloliquefaciens CH1:
During fermentation condition optimization, adopt the Plackett-Burman design from 11 factors, to select earlier lipopeptid output is had the remarkable sex factor, respectively the important factor of selecting is done experiment of single factor again and be optimized, utilize field mouth design to determine final medium component and each factor level at last.With the bacteriostasis rate to oil tea anthrax is unique response amount of fermentation test.
Bacteriostasis rate calculates: seed liquor is cultivated 12h in the PD substratum after, be equipped with in the 250mL triangular flask of 50mL fermention medium with the access of 5% inoculum size, fermentation 48h, 8000rmp, centrifugal 5min, collect supernatant liquor and with after the 0.22 μ m membrane filtration degerming, mixes with 1: 50 volume ratio with the PDA substratum that to be inverted flat board standby.Will be in the PDA flat board cultured oil tea anthrax bacteria, buy the bacterium cake that cut-off directly is 0.5cm with punch tool, the bacterium cake is inoculated in the PDA flat board of the above-mentioned CH1 of containing filtration sterilization fermented liquid, be seeded on the PDA flat board that does not contain the CH1 fermented liquid with the bacterium cake and do contrast.Be inverted for 28 ℃ and cultivate, wait to contrast when mycelia is soon covered with plate in the plate, the colony diameter of experiment with measuring group and control group calculates bacteriostasis rate.
Bacteriostasis rate (%)=(control group diameter-experimental group diameter)/(control group diameter-0.5) * 100%
(1) Plackett-Burman design:
9 medium components (bean cake powder, Semen Maydis powder, starch, the NH that might influence lipopeptid output
4NO
3, MgSO
4, yeast powder, KH
2PO
4, Na
2HPO
4, CaCl
2), (pH6.0 pH7.5) carries out Plackett-Burman as test factor and designs together with leavening temperature (30 ℃, 37 ℃) and fermentation pH value.The growth of thalline and the generation of lipopeptid need nutritive substances such as carbon source, nitrogenous source, inorganic salt and trace element, in the design, comprise Semen Maydis powder, starch, yeast powder for the examination carbon source; Nitrogenous source comprises organic nitrogen source bean cake powder and inorganic nitrogen-sourced ammonium nitrate; Necessary nutritive substance (the NH of biological growth metabolic process such as magnesium elements, sodium element, phosphoric and calcium constituent have also been added
4NO
3, MgSO
4, KH
2PO
4, Na
2HPO
4, CaCl
2); Because initial medium contains a large amount of organism and dissolve medium is a tap water, do not consider the extra trace element (Fe, Cu, Zn, Mn, Co etc.) that adds.
(2) experiment of single factor:
Result according to the Plackett-Burman design, deletion is to lipopeptid output does not make significant difference and consumption is less medium component, selection has the factor of remarkably influenced to be optimized with experiment of single factor respectively to lipopeptid output, in single factor optimizing process, variation according to the response amount, constantly dwindle the optimization range of substratum concentration, to find optimum substratum concentration, at last to Na
2HPO
4, NH
4NO
3Be optimized with following concentration respectively with bean cake powder.
Na
2HPO
4Output is to the influence of lipopeptid output: on the screening basis of Plackett-Burman design, fix other components unchanged, with different Na
2HPO
4Concentration: 0g/L, 4g/L, 8g/L, 12g/L experimentizes, and calculates the fermented liquid bacteriostasis rate, determines Na
2HPO
4Suitable concn (4g/L).
NH
4NO
3Concentration is to the influence of lipopeptid output: on the screening basis of Plackett-Burman design, fix other components unchanged, with different NH
4NO
3Concentration: 0g/L, 4g/L, 8g/L, 12g/L, 16g/L experimentizes, and calculates the fermented liquid bacteriostasis rate, determines NH
4NO
3Suitable concn (4g/L).
Bean cake powder concentration is to the influence of lipopeptid output: on the screening basis of Plackett-Burman design, fix other components unchanged, with different bean cake powder concentration: 4g/L, 8g/L, 12g/L, 16g/L, 20g/L, 24g/L experimentizes, and calculates the fermented liquid bacteriostasis rate, determines the suitable concn (12g/L) of bean cake powder.
(3) design is tested in the field cause for gossip
According to the optimum result of experiment of single factor, select Na
2HPO
4, NH
4NO
3, the starch of bean cake powder and large usage quantity is totally 4 factors, respectively establishes 3 levels, carries out L9 (3)
4The field oral examination is tested.According to the field oral examination test the result to the higher substratum of output combination verify.
Embodiment 2:
The application of a kind of bacillus amyloliquefaciens of fungus apoptosis lipopeptide in preparation treatment or prevention oil tea anthrax medicine, its applying step is:
1, bacillus amyloliquefaciens CH1 is to the inhibition activity of oil tea anthrax:
(1) no fermented liquid obtains:
Bacillus amyloliquefaciens CH1 bacterial classification (being separated to rice root) the 10 μ L that inoculation is preserved in the PD substratum of 10mL sterilization, 37 ℃, 200rpm shaking culture 12h.Inoculate 5mL fresh culture thing then in the 250mL triangular flask that contains the fresh PD substratum of 100mL, 30 ℃, 200rpm shaking culture 48h, the centrifugal 10min of 8000g collects supernatant, and supernatant liquor-20 ℃ of preservations after the sterilization of 0.22 μ m membrane filtration are standby.
(2) the CH1 fermented liquid is to the inhibition of oil tea anthrax mycelia:
CH1 is not had fermented liquid and PDA substratum, and to be that 1: 50,1: 20,1: 10 mixed is fallen respectively by volume dull and stereotyped standby, with the PDA substratum that do not add fermented liquid in contrast.Oil tea anthrax spore is diluted to 1 * 10 with sterilized water
5/ mL gets 200 μ L spread plates, is inverted for 37 ℃ and cultivates 24h.Get the mycelia compressing tablet, microscopically is observed the mycelia form.
(3) the CH1 fermented liquid on excised leaf to the inhibition of oil tea anthrax:
Gather fresh oil tea tender leaf of the same size, be placed in the culture dish that is covered with 1 layer of wet filter paper after clean, cover at the petiole place with the water-moistened absorbent cotton of distillation with distilled water flushing.Test group is sprayed blade with the no fermented liquid of 50 times of distilled water dilutings, 1 oil tea anthrax bacteria cake of inoculation after room temperature (20-25 ℃, below the identical) seasoning; The blade of control group sprays 1 oil tea anthrax bacteria cake of inoculation after the distilled water drying, and 2 groups place temperature simultaneously is 26 ℃, and humidity is to cultivate in 95% the illumination box, and every 24h observes 1 time and also adds an amount of distilled water and preserve moisture to absorbent cotton.
2, the preparation of bacillus amyloliquefaciens CH1 spray-dried powders:
The amplification of fermenting process is carried out in 3L, 20L, 500L fermentor tank successively, and oxygen dissolving value is measured by dissolved oxygen electrode, and the pH value adopts substratum nature pH value (pH7.0).Major control dissolved oxygen and stirring revolution between yeast phase, ventilation is initially stirred revolution 200rpm than 0.5～1.5vvm, keeps dissolved oxygen more than 10% in the fermenting process, and culture temperature is constant in 30 ℃, adds defoamer elimination foam after foam forms.
When using the 3L fermentor tank to ferment, the 50mL seed liquor inserts in the 3L fermentor tank that the 1.5L substratum is housed ferments, and fermentation surpasses after the 12h, and every 3h sampling once.Sample is removed thalline and fermentation residue through centrifugal and aseptic membrane filtration, detects its restraining effect to oil tea anthrax respectively, to understand the generation period of lipopeptid.
After fermentation was finished, through 65 ℃, 0.09MPa concentrated fermented liquid, with 25% of fermented liquid simmer down to original volume with the concentrating under reduced pressure jar.Fermented liquid after concentrating uses spray-drying tower with 160 ℃ of intake air temperatures, 80 ℃ of air outlet temperature, and the condition of feed liquor amount 10mL/min is carried out spray drying treatment.Spray-dried powders is carried out weighing, calculate powder output.
According to spray-dried powders output, be mixed with and the solution of fermented liquid same concentrations with the CH1 powder without spraying, with 0.22 μ m membrane filtration degerming after, detection solution bacteriostasis rate.Same method detects the CH1 powder fungistatic effect in 1 year of room temperature preservation.
3, CH1 spray-dried powders control oil tea anthrax nursery experiment:
The test base is positioned at Zheng Dian woods fruit base, Jiangxia, and the oil tea seedling is 2 years living cuttage seeding.3 processing are established in test altogether: 1000 times of liquid of bacillus amyloliquefaciens CH1 wettable powder; 600 times of liquid of 50% (w/w) carbendazol wettable powder; 1000 times of liquid of 70% (w/w) thiophanate methyl wettable powder; Other establishes and sprays clear water as blank.4 repetitions, totally 16 processing are established in each processing.
The processing of spraying of oil tea their early stage is carried out dispenser once every 10d later on, continuous use 5 times.Carry out the investigation first time behind medication 15d, 10d carries out the investigation second time after the last medication.
The investigation of tea shoot incidence is a unit with the plant, the random sampling of every sub-district, and total strain number and diseased plant number that record is spot-check calculate sickness rate.According to the disease grade scale, investigation oil tea blade incidence in investigate morbidity strain, record blade amt and blade occurring degree calculate disease index.
Grade scale:
0 grade: blade does not have scab;
1 grade: scab accounts for the blade total area less than 5%;
3 grades: scab accounts for blade total area 6-10%;
5 grades: scab accounts for blade total area 11-25%;
7 grades: scab accounts for blade total area 26-50%;
9 grades: scab accounts for the blade total area more than 50%.
The drug effect method of calculation:
The total strain number of sickness rate=morbidity strain tree/investigation * 100%
Disease index=∑ (the sick numbers of sheets at different levels * relative level numerical value)/(investigation sum * 9) * 100%.
Claims (3)
1. the isolating product of a strain is urged the bacillus amyloliquefaciens bacterial strain of fungus apoptosis lipopeptide, it is characterized in that: bacillus amyloliquefaciens (
Bacillus amyloliquefaciens) CH1, CCTCC:M208127.
2. the preparation method of the bacillus amyloliquefaciens of the described a kind of fungus apoptosis lipopeptide of claim 1 the steps include:
The screening and separating of A, bacillus amyloliquefaciens: the paddy rice of gathering tillering phase from rice terrace, the rice root outside surface is rinsed well with tap water, again with aseptic water washing 2-4 time, then with 70%v/v alcohol immersion sterilization 28-32s, behind aseptic water washing 2-4 time, rice root is cut into the fragment of 1mm size, places the PDA substratum: contain the 200g potato and cook liquid, 20g glucose, 20g agar, adding distil water is gone up in 28 ℃ of cultivation 46-50h to 1L, obtains single bacterium colony;
The bacteriostatic activity of B, bacterial detection: the bacterium that separation is obtained is with the PD liquid nutrient medium: contain the 200g potato and cook liquid, 20g glucose, adding distil water is to 1L, in 28 ℃, shaking culture, fermented supernatant fluid with 0.22 μ m membrane filtration after, mix the Rhizoctonia solani Kuhn of falling the plating by volume with the PDA substratum, detect anti-microbial activity;
The classification of C, bacterium is identified: the bacterium that detects remarkable anti-mycotic activity is carried out 16SrRNA and identification of morphology;
Extract the genomic dna of bacterium with ordinary method, carry out PCR with the universal primer of 16SrRNA, amplified production carries out being defined as bacillus amyloliquefaciens through the Blast comparative analysis behind the sequencing in NCBI;
D, microbial culture and store method: the condition of cultivating bacillus amyloliquefaciens is PD substratum or LB substratum, 28 ℃ of-37 ℃ of cultivations, and bacterial strain is preserved: get cultured
Bacillus.
AmyloliquefaciensCH1 bacterium liquid add sterile glycerol to final concentration 25% in-80 ℃ of preservations.
3. the application of the bacillus amyloliquefaciens of the described a kind of fungus apoptosis lipopeptide of claim 1 in preparation treatment or prevention oil tea anthrax medicine.
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